We will develop technologies for the [unreadable]Structural Restraints[unreadable] and [unreadable]Structure Calculation[unreadable] components of the pipeline proposed in Fig. 3 above, and apply them to determine the structures of mitochondrial translocators. In addition to their primary role of ATP synthesis, mitochondria house several basic biological processes, such as generation of reactive oxygen species, apoptosis, thermogenesis, and biosynthesis of cholesterol and steroid hormones. Metabolites and ions move selectively in and out of mitochondria through membrane-embedded translocators. As expected from their roles of translocating substrates, these proteins must constantly switch between multiple conformational states, which is probably why they are difficult targets to crystallize. We will solve the structures of three important membrane translocators: the 30 kDa GDP/GTP carrier (GGC) and uncoupling protein 2 (UCP2) of the inner mitochondrial membrane, and the 19 kDa peripheral benzodiazepine receptor (PBR) of the outer membrane. GGC and UCP2 are members of the large mitochondrial carrier family that translocates purine nucleotides and protons across the inner membrane, respectively. PBR is involved in the steroidogenesis-limiting import of cholesterol across the outer membrane. These proteins are all polytopic helical MPs for which structures are not known. Polytopic helical MPs pose different problems of assigning long-range NOEs than water-soluble proteins because the two types of proteins have different folding properties in terms of packing of amino acids (AA) in the protein core. The former also has the problem of strong resonance overlap in both backbone and sidechain resonances. We will develop methods and protocols in two technical areas to solve these problems. First, we will test various isotope labeling schemes and evaluate their effectiveness for measuring inter-helical NOEs by using high resolution 4D NOE experiments to be developed by Wagner (Project 2). Second, we will establish robust protocols for obtaining global orientation restraints from residual dipolar couplings (RDCs). We will work with Core C to integrate the new technologies into the proposed NMR pipeline.